Detection, target amplification and sequencing of nucleic acids are pivotal methods in molecular biology, in research as well as in clinical diagnostics, and key reagents in such methods are oligonucleotides acting as primers and/or probes.
Of main importance for primers and probes are their sequence specificity and also their affinity for a complementary nucleic acid. These features can be modulated by factors intrinsic to the oligonucleotide and factors extrinsic to the oligonucleotide. Intrinsic factors are e.g. the length and nucleic acid sequence composition of oligonucleotides. Also the uses of non-natural nucleotides or backbone modifications are intrinsic factors. However, the number of available non-natural nucleotides and backbone units are limited. Accordingly, there is a need for oligonucleotides with novel modifications that can be used in molecular biology methods.
Patent application WO 2006/125447 described a triplex forming monomer unit of the formula Z (described below) and demonstrated favorable characteristics of an oligonucleotide comprising a triplex forming monomer unit with regards to triplex formation with a double stranded nucleic acid. Based on the triplex forming characteristics, the inventors of the aforementioned patent application suggest using the oligonucleotide for detection, diagnosis and treatment. No details or data on such uses were provided.
Filichev at al., (Filichev V V, 2005) described the same triplex forming monomer unit as WO 2006/125447 and found stabilization of parallel duplex and parallel triplex by incorporation of the triplex forming monomer unit. Moreover, they found destabilization of Watson-Crick type RNA/DNA and DNA/DNA duplexes when triplex forming monomer units were inserted into an oligonucleotide, compared to the native oligonucleotide.